The overall goal of this project is to exploit the RNA interference (RNAi) mechanism in order to develop therapeutics to treat human disease. Because the RNAi mechanism is extraordinarily potent in reducing expression of a specific gene, it offers an opportunity to rationally design drugs targeted to individual genes in a disease implicated pathway. While this paradigm has largely been realized in cell culture using cationic lipids to introduce substrates for the RNAi pathway into mammalian cells, the widespread application of RNAi technology to gene inhibition in animals remains elusive. This is largely due to the poor pharmacokinetic (PK) properties of RNAi based drugs such as small interfering RNAs (siRNAs). We believe that that unmodified siRNA will fail as systemically administered drugs due to the poor PK properties of RNA, in particular insufficient stability in biological fluids such as blood, and suboptimal uptake by tissues and penetration into cells. We intend to overcome these limitations by employing our knowledge of the structure-activity relationships (SAR) of RNAi drugs to design analogs which have the potential to exhibit improved PK properties, yet maintain the high intrinsic potency of the RNAi mechanism. We will then evaluate these improved designs in preliminary pharmacokinetic and pharmacology studies in order to assess their ability to serve as leads for the development of robust RNAi based therapeutics. The deliverable from the project is one or more chemically modified RNAi constructs which show superior pharmacokinetics and robust pharmacology in animal models. Because of our focus on a novel target relevant to cancer chemotherapy, we will also generate therapeutic leads appropriate for pre-clinical drug development activities. The deliverables from the project will be optimized chemically modified motifs for RNAi drugs which should be widely applicable to the modulation of gene expression in animals as well as a lead siRNA compound targeting Eg5 that will be ready to begin preclinical drug development activities.